Plasticity in Skeletal, Cardiac, and Smooth Muscle: Selected Contribution: IGF-I antibody prevents increases in protein synthesis in epitrochlearis muscles from refed, diabetic rats

The purpose of this study was to examine whether immune neutralizationof muscle-produced insulin-like growth factor I (IGF-I) would preventan appropriate anabolic response to refeeding in diabetic rats. MaleSprague-Dawley rats were made diabetic by partial pancreatectomy andwere randomly assigned to be either control-fed, fasted, orfasted-refed (n = 7-8 per group). Diabetes decreased rates of protein synthesis and increased rates of protein degradation in incubated epitrochlearis muscles (P < 0.05). In both groups of rats, fasting lowered protein synthesis andincreased proteolysis and subsequent refeeding returned both parameters to near basal values (P < 0.05). Neutralization ofmuscle IGF-I by the addition of IGF-I antibody to the incubation mediumreduced protein synthesis an average of 22% for all groups(P < 0.05). However, rates of protein degradation werenot affected. In nondiabetic rats, refeeding increased proteinsynthesis in both control and antibody-treated muscles(P < 0.05). Refeeding also increased protein synthesisin the control muscles from diabetic rats (P < 0.01).In contrast, muscles from diabetic rats that were incubated withanti-IGF-I did not increase protein synthesis in response to refeeding.These data suggest that immune neutralization of muscle IGF-I inhypoinsulinemic rats negated the ability of endogenous IGF-I to promoteprotein synthesis and thereby prevented an appropriate anabolic response.

     

Immunization against IGF-I prevents increases in protein synthesis in diabetic rats after resistance exercise

These studies examined whether passive immunization against insulin-like growth factor I (IGF-I) would prevent increases in rates of protein synthesis in skeletal muscle of diabetic rats after resistance exercise. Male Sprague-Dawley rats were pancreatectomized and randomly assigned to either an exercise or a sedentary group. Animals in each of these groups received either an IGF-I antibody or a nonspecific IgG from a subcutaneous osmotic pump. Exercise did not change plasma or gastrocnemius IGF-I concentrations in nondiabetic rats. However, plasma and muscle IGF-I concentrations were higher in IgG-treated diabetic rats that exercised compared with respective sedentary groups (P < 0.05). Passively immunized diabetic rats did not exhibit the same exercise-induced increase in IGF-I concentrations. In nondiabetic rats, protein synthesis rates were higher after exercise in both control and immunized groups. In diabetic rats, exercise increased protein synthesis in the IgG-treated animals but not in those treated with IGF-I antibody. There was also a significant positive correlation between both plasma and gastrocnemius IGF-I concentrations and rates of protein synthesis in diabetic (P < 0.01), but not nondiabetic, rats. These results suggest that IGF-I is compensatory for insulin in hypoinsulinemic rats by facilitating an anabolic response after acute resistance exercise.     

Active involvement of PKC for insulin-mediated rates of muscle protein synthesis in Zucker rats

A recent report from our group demonstrated that insulin facilitates muscle protein synthesis in obese Zucker rats. The purpose of this study was to determine whether PKC, a probable modulator of insulin signal transduction and/or mRNA translation, has a role in this insulin-mediated anabolic response. In the first portion of the study, gastrocnemius muscles of lean and obese Zucker rats (n = 5-7 for each phenotype) were bilaterally perfused with or without insulin to assess cytosolic and membrane PKC activity. Limbs perfused with insulin demonstrated greater PKC activity in both lean and obese Zucker rats (P < 0.05) compared with no insulin, but overall activity was greater in obese animals (by approximately 27% compared with lean, P < 0.05). To determine whether PKC plays a role in muscle protein synthesis, hindlimbs (n = 6-8 for each phenotype) were bilaterally perfused with or without insulin and/or GF-109203X (GF; a PKC inhibitor). The presence of GF did not influence the rates of insulin-mediated protein synthesis in gastrocnemius muscle of lean Zucker rats. However, when obese rats were perfused with GF (P < 0.05), the effect of insulin on elevating rates of protein synthesis was not observed. We also used phorbol 12-myristate 13-acetate (TPA, a PKC activator; n = 5-7 for each phenotype) with and without insulin to determine the effect of PKC activation on muscle protein synthesis. TPA alone did not elevate muscle protein synthesis in lean or obese rats. However, TPA plus insulin resulted in elevated rates of protein synthesis in both phenotypes that were similar to rates of insulin alone of obese rats. These results suggest that PKC is a modulator and is necessary, but not sufficient, for insulin-mediated protein anabolic responses in skeletal muscle.     

The effect of glutamine on protein turnover in chick skeletal muscle in vitro.

The effect of glutamine on the rates of protein synthesis and degradation was studied in isolated chick extensor digitorum communis muscles incubated in the presence of plasma concentrations of amino acids. Addition of 0.5-15 mM-glutamine increases (P less than 0.01) intracellular glutamine concentrations by 31-670%. There is a positive relationship (r = 0.975, P less than 0.01) between intracellular glutamine concentration and the rate of muscle protein synthesis measured by the incorporation of [3H]phenylalanine. The stimulating effect of 15 mM-glutamine on protein synthesis was decreased from 58 to 19% in muscles incubated in the absence of tyrosine. The rates of protein degradation, estimated from [3H]phenylalanine release from muscle proteins prelabelled in vivo, decreased (P less than 0.05) by 15-30% in the presence of 4-15 mM-glutamine when compared with muscles incubated in the presence of physiological concentrations of glutamine (0.5-1 mM). Glutamine concentrations ranging from 2 to 15 mM appear to have an overall anabolic effect on chick skeletal muscles incubated in vitro.     

Insulin facilitation of muscle protein synthesis following resistance exercise in hindlimb-suspended rats is independent of a rapamycin-sensitive pathway

Hindlimb suspension (HS) results in rapid losses of muscle mass, which may in part be explained by attenuated rates of protein synthesis. Mammalian target of rapamycin (mTOR) regulates protein synthesis and has been implicated as a potential mediator of the muscle mass decrement with HS. This study examined the effect of resistance exercise, a muscle hypertrophy stimulant, on rates of protein synthesis after 4 days of HS in mature male Sprague-Dawley rats. Flywheel resistance exercise (2 sets x 25 repetitions) was conducted on days 2 and 4 of HS (HSRE). Sixteen hours after the last exercise bout, soleus muscles were assessed for in vitro rates of protein synthesis, with and without insulin (signaling agonist) and/or rapamycin (mTOR inhibitor). Results demonstrated that soleus mass was reduced (P < 0.05) with HS, but this loss of mass was not observed (P > 0.05) with HSRE. Muscle protein synthesis was diminished (P < 0.05) with HS, with or without insulin. HSRE also had reduced rates of synthesis without insulin; however, insulin administration yielded higher (P < 0.05) rates in HSRE compared with HS or control. Rapamycin diminished protein synthesis in all groups (P < 0.05), but insulin rescued synthesis rates in HS and HSRE to levels similar to insulin alone for each group, suggesting that alternate signaling pathways develop to increase protein synthesis with HS. These results demonstrate that the capacity for an augmented anabolic response to resistance exercise is maintained after 4 days of HS and is independent of a rapamycin-sensitive pathway.     

Skeletal muscle protein synthesis and the abundance of the mRNA translation initiation repressor PDCD4 are inversely regulated by fasting and refeeding in rats

Optimal skeletal muscle mass is vital to human health, because defects in muscle protein metabolism underlie or exacerbate human diseases. The mammalian target of rapamycin complex 1 is critical in the regulation of mRNA translation and protein synthesis. These functions are mediated in part by the ribosomal protein S6 kinase 1 (S6K1) through mechanisms that are poorly understood. The tumor suppressor programmed cell death 4 (PDCD4) has been identified as a novel substrate of S6K1. Here, we examined 1) the expression of PDCD4 in skeletal muscle and 2) its regulation by feed deprivation (FD) and refeeding. Male rats (~100 g; n = 6) were subjected to FD for 48 h; some rats were refed for 2 h. FD suppressed muscle fractional rates of protein synthesis and Ser(67) phosphorylation of PDCD4 (-50%) but increased PDCD4 abundance (P < 0.05); refeeding reversed these changes (P < 0.05). Consistent with these effects being regulated by S6K1, activation of this kinase was suppressed by FD (-91%, P < 0.05) but was increased by refeeding. Gavaging rats subjected to FD with a mixture of amino acids partially restored muscle fractional rates of protein synthesis and reduced PDCD4 abundance relative to FD. Finally, when myoblasts were grown in amino acid- and serum-free medium, phenylalanine incorporation into proteins in cells depleted of PDCD4 more than doubled the values in cells with a normal level of PDCD4 (P < 0.0001). Thus feeding stimulates fractional protein synthesis in skeletal muscle in parallel with the reduction of the abundance of this mRNA translation inhibitor.     

Skeletal and cardiac muscle protein turnover during cold acclimation in young rats

The effect of long-term cold exposure on skeletal and cardiac muscle protein turnover was investigated in young growing animals. Two groups of 36 male 28-day-old rats were maintained at either 5 degrees C (cold) or 25 degrees C (control). Rates of protein synthesis and degradation were measured in vivo on days 5, 10, 15, and 20. Protein mass by day 20 was approximately 28% lower in skeletal muscle (gastrocnemius and soleus) and approximately 24% higher in heart in cold compared with control rats (P < 0.05). In skeletal muscle, the fractional rates of protein synthesis (k(syn)) and degradation (k(deg)) were not significantly different between cold and control rats, although k(syn) was lower (approximately -26%) in cold rats on day 5; consequent to the lower protein mass, the absolute rates of protein synthesis (approximately -21%; P < 0. 05) and degradation (approximately -13%; P < 0.1) were lower in cold compared with control rats. In heart, overall, k(syn) (approximately +12%; P < 0.1) and k(deg) (approximately +22%; P < 0.05) were higher in cold compared with control rats; consequently, the absolute rates of synthesis (approximately +44%) and degradation (approximately +54%) were higher in cold compared with control rats (P < 0.05). Plasma triiodothyronine concentration was higher (P < 0.05) in cold compared with control rats. These data indicate that long-term cold acclimation in skeletal muscle is associated with the establishment of a new homeostasis in protein turnover with decreased protein mass and normal fractional rates of protein turnover. In heart, unlike skeletal muscle, rates of protein turnover did not appear to immediately return to normal as increased rates of protein turnover were observed beyond day 5. These data also indicate that increased rates of protein turnover in skeletal muscle are unlikely to contribute to increased metabolic heat production during cold acclimation.     

Classification of Chitosanases by Hydrolytic Specificity toward N 1,N 4-Diacetylchitohexaose

The immediate response of protein degradation to food intake and the factors for its regulation in rat skeletal muscle were examined. The concentration of N ^τ-methylhistidine (MeHis) in serum and the rates of MeHis release from isolated soleus and extensor digitorum longus muscles were reduced in the period from 3 to 6h after refeeding, indicating that the rate of myofibrillar protein degradation in the rat decreased immediately after refeeding. Changes in the serum concentration of insulin and corticosterone were not synchronized with those in the myofibrillar protein degradation. When rats were fed on a protein-free diet, no reduction of serum MeHis concentration or of the rate of MeHis release from isolated muscles after refeeding was apparent. Furthermore, there was a tendency toward suppressing myofibrillar protein degradation with a higher protein content of the diet. These results suggest that the suppression of myofibrillar protein degradation by food intake was regulated by dietary proteins.     

Higher skeletal muscle protein synthesis and lower breakdown after chemotherapy in cachectic mice

The influence of cancer cachexia and chemotherapy and subsequent recovery of skeletal muscle protein mass and turnover was investigated in mice. Cancer cachexia was induced using colon 26 adenocarcinoma, which is characteristic of the human condition, and can be cured with 100% efficacy using an experimental nitrosourea, cystemustine (C(6)H(12)CIN(3)O(4)S). Reduced food intake was not a factor in these studies. Three days after cachexia began, healthy and tumor-bearing mice were given a single intraperitoneal injection of cystemustine (20 mg/kg). Skeletal muscle mass in tumor-bearing mice was 41% lower (P < 0.05) than in healthy mice 2 wk after cachexia began. Skeletal muscle wasting was mediated initially by decreased protein synthesis (-38%; P < 0.05) and increased degradation (+131%; P < 0.05); later wasting resulted solely from decreased synthesis (~-54 to -69%; P < 0.05). Acute cytotoxicity of chemotherapy did not appear to have an important effect on skeletal muscle protein metabolism in either healthy or tumor-bearing mice. Recovery began 2 days after treatment; skeletal muscle mass was only 11% lower than in healthy mice 11 days after chemotherapy. Recovery of skeletal muscle mass was affected initially by decreased protein degradation (-80%; P < 0.05) and later by increased protein synthesis (+46 to +73%; P < 0.05) in cured compared with healthy mice. This study showed that skeletal muscle wasted from cancer cachexia and after chemotherapeutic treatment is able to generate a strong anabolic response by making powerful changes to protein synthesis and degradation.     

Studies concerning the specificity of the effect of leucine on the turnover of proteins in muscles of control and diabetic rats.

The protein anabolic effect of branched chain amino acids was studied in isolated quarter diaphragms of rats. Protein synthesis was estimated by measuring tyrosine incorporation into muscle proteins in vitro. Tyrosine release during incubation with cycloheximide served as an index of protein degradation. In muscles from normal rats the addition of 0.5 mM leucine stimulated protein synthesis 36--38% (P less than 0.01), while equimolar isoleucine or valine, singly or in combination were ineffective. The three branched chain amino acids together stimulated no more than leucine alone. The product of leucine transamination, alpha-keto-isocaproate, did not stmino norborane-2-carboxylic acid (a leucine analogue) were ineffective. Leucine and isoleucine stimulated protein synthesis in muscles from diabetic rats.Leucine, isoleucine, valine and the norbornane amino acid but not alpha-ketoisocaproate or beta-hydroxybutyrate decreased the concentration of free tyrosine in tissues during incubation with cycloheximide; tyrosine release into the medium did not decrease significantly. Leucine caused a small decrease in total tyrosine release, (measured as the sum of free tyrosine in tissues and media), suggesting inhibition of protein degradation. The data suggest that leucine may be rate limiting for protein synthesis in muscles. The branched chain amino acids may exert a restraining effect on muscle protein catabolism during prolonged fasting and diabetes.     

Acute alcohol administration inhibits the refeeding response after starvation in rat skeletal muscle

This study determined whether an acute alcohol dose could inhibit the refeeding response in starved muscle. Rats starved for 24 h were pretreated with alcohol or saline before refeeding by intragastric or intravenous infusion of enteral diet (ENT), total parenteral nutrition (TPN), or saline. Refeeding by TPN or ENT stimulated increases in the fractional rate of protein synthesis (k(s)) in skeletal muscle. Alcohol prevented the increase in k(s) when refeeding occurred intragastrically (TPN or ENT) (P < 0.001) but not intravenously (TPN). Upon intragastric refeeding, alcohol inhibited the increase in both eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) and p70 S6 kinase (p70(S6K)) phosphorylation in plantaris but caused only partial inhibition in soleus muscle (ENT only). When rats were refed intravenously, alcohol had no effect on the increased 4E-BP1 or p70(S6K) phosphorylation in either muscle. Plasma insulin levels were augmented by alcohol. Alcohol-related changes in plasma amino acid concentrations were similar irrespective of the route of feeding, whereas IGF-I levels showed differential changes. This is the first study to demonstrate that acute alcohol ingestion impedes the starved-to-fed response in skeletal muscle.     

Pentoxifylline inhibits Ca2+-dependent and ATP proteasome-dependent proteolysis in skeletal muscle from acutely diabetic rats

Previous studies from this laboratory have shown that catecholamines exert an inhibitory effect on muscle protein degradation through a pathway involving the cAMP cascade. The present work investigated the systemic effect of pentoxifylline (PTX; cAMP-phosphodiesterase inhibitor) treatment on the rate of overall proteolysis, the activity of proteolytic systems, and the process of protein synthesis in extensor digitorum longus muscles from normal and acutely diabetic rats. The direct in vitro effect of this drug on the rates of muscle protein degradation was also investigated. Muscles from diabetic rats treated with PTX showed an increase (22%) in the cAMP content and reduction in total rates of protein breakdown and in activity of Ca2+-dependent (47%) and ATP proteasome-dependent (23%) proteolytic pathways. The high content of m-calpain observed in muscles from diabetic rats was abolished by PTX treatment. The addition of PTX (10(-3) M) to the incubation medium increased the cAMP content in muscles from normal (22%) and diabetic (51%) rats and induced a reduction in the rates of overall proteolysis that was accompanied by decreased activity of the Ca2+-dependent and ATP proteasome-dependent proteolytic systems, in both groups. The in vitro addition of H-89, an inhibitor of protein kinase A (PKA), completely blocked the effect of PTX on the reduction of proteolysis in muscles from normal and diabetic rats. The present data suggest that PTX exerts a direct inhibitory effect on protein degradative systems in muscles from acutely diabetic rats, probably involving the participation of cAMP intracellular pathways and activation of PKA, independently of tumor necrosis factor-alpha inhibition.     

Comparison of protein synthesis and degradation in incubated and perfused muscle.

Rates of muscle protein synthesis and degradation measured in the perfused hindquarter were compared with those in incubated epitrochlearis muscles. With fed or starved mature rats, results without insulin treatment were identical. With insulin treatment, protein synthesis in perfused hindquarters was greater, though protein degradation was the same. Thus rates of muscle protein degradation estimated by these two methods in vitro correspond closely.     

Rapid suppression of protein degradation in skeletal muscle after oral feeding of leucine in rats

A diet containing adequate amounts of protein rapidly suppresses myofibrillar protein degradation in rats and mice. This study determined whether dietary amino acids inhibit postprandial protein degradation in rat skeletal muscle. When rats fed on a 20% casein diet for 1 h after 18 h starvation, the rate of myofibrillar protein degradation measured by N(tau)-methylhistidine release from the isolated extensor digitorum longus muscle was significantly (p < 0.05) decreased at 4 h after refeeding. A diet containing an amino acid mixture which is the same composition as casein also reduced myofibrillar protein degradation at 4 h after refeeding (p < 0.05). An essential amino acid mixture (15.1%, corresponding to casein composition) and a leucine (2.9%) diets reduced the rate of myofibrillar protein degradation after refeeding (p < 0.05), whereas a protein free diet did not. Administration of leucine alone (0.135 g/100 g body weight) by a feeding tube induced a decrease in the rate of myofibrillar protein degradation at 2 h after administration (p < 0.05), whereas the serum insulin concentration was constant after leucine administration. These results suggested that leucine is one of regulating factors of myofibrillar protein degradation after refeeding of a protein diet.     

The effects of denervation on protein turnover of the soleus and extensor digitorum longus muscles of adult mice.

1. Changes in protein turnover of the soleus and EDL muscles of adult mice have been studied 1, 7 and 80 days after denervation. 2. Increased rates of protein degradation 7 and 80 days post-denervation correlated with the atrophy and loss of protein from these muscles. 3. Rates of protein synthesis in the EDL decreased 24 hr after nerve section. However, these synthetic rates increased again to become higher in the 7 day denervated muscles compared with their controls. These latter anabolic changes are inconsistent with the concept of a denervated muscle being inactive. 4. These findings have been compared with a similar study on muscles of growing rats. Any passive stretching of the denervated muscles by continued bone growth appears unlikely to be a crucial factor explaining the increased rates of protein synthesis 7 days after denervation.     

Testosterone increases the muscle protein synthesis rate but does not affect very-low-density lipoprotein metabolism in obese premenopausal women

Men and women with hyperandrogenemia have a more proatherogenic plasma lipid profile [e.g., greater triglyceride (TG) and total and low-density lipoprotein-cholesterol and lower high-density lipoprotein-cholesterol concentrations] than healthy premenopausal women. Furthermore, castration of male rats markedly reduces testosterone availability below normal and decreases plasma TG concentration, and testosterone replacement reverses this effect. Testosterone is, therefore, thought to be an important regulator of plasma lipid homeostasis. However, little is known about the effect of testosterone on plasma TG concentration and kinetics. Furthermore, testosterone is a potent skeletal muscle protein anabolic agent in men, but its effect on muscle protein turnover in women is unknown. We measured plasma lipid concentrations, hepatic very low density lipoprotein (VLDL)-TG and VLDL-apolipoprotein B-100 secretion rates, and the muscle protein fractional synthesis rate in 10 obese women before and after trandermal testosterone (1.25 g of 1% AndroGel daily) treatment for 3 wk. Serum total and free testosterone concentrations increased (P < 0.05) by approximately sevenfold in response to testosterone treatment, reaching concentrations that are comparable to those in women with hyperandrogenemia, but lower than the normal range for eugonadal men. Except for a small (～10%) decrease in plasma high-density lipoprotein particle and cholesterol concentrations (P < 0.04), testosterone therapy had no effect on plasma lipid concentrations, lipoprotein particle sizes, and hepatic VLDL-TG and VLDL-apolipoprotein B-100 secretion rates (all P > 0.05); the muscle protein fractional synthesis rate, however, increased by ～45% (P < 0.001). We conclude that testosterone is a potent skeletal muscle protein anabolic agent, but not an important regulator of plasma lipid homeostasis in obese women.     

Severe diabetes prohibits elevations in muscle protein synthesis after acute resistance exercise in rats.

This study determined whether rates of protein synthesis increase after acute resistance exercise in skeletal muscle from severely diabetic rats. Previous studies consistently show that postexercise rates of protein synthesis are elevated in nondiabetic and moderately diabetic rats. Severely diabetic rats performed acute resistance exercise (n = 8) or remained sedentary (n = 8). A group of nondiabetic age-matched rats served as controls (n = 9). Rates of protein synthesis were measured 16 h after exercise. Plasma glucose concentrations were >500 mg/dl in the diabetic rats. Rates of protein synthesis (nmol phenylalanine incorporated. g muscle(-1). h(-1), means +/- SE) were not different between exercised (117 +/- 7) and sedentary (106 +/- 9) diabetic rats but were significantly (P < 0.05) lower than in sedentary nondiabetic rats (162 +/- 9) and in exercised nondiabetic rats (197 +/- 7). Circulating insulin concentrations were 442 +/- 65 pM in nondiabetic rats and 53 +/- 11 and 72 +/- 19 pM in sedentary and exercised diabetic rats, respectively. Plasma insulin-like growth factor I concentrations were reduced by 33% in diabetic rats compared with nondiabetic rats, and there was no difference between exercised and sedentary diabetic rats. Muscle insulin-like growth factor I was not affected by resistance exercise in diabetic rats. The results show that there is a critical concentration of insulin below which rates of protein synthesis begin to decline in vivo. In contrast to previous studies using less diabetic rats, severely diabetic rats cannot increase rates of protein synthesis after acute resistance exercise.     

Ghrelin receptor agonist GHRP-2 prevents arthritis-induced increase in E3 ubiquitin-ligating enzymes MuRF1 and MAFbx gene expression in skeletal muscle

Chronic arthritis is a catabolic state associated with an inhibition of the IGF system and a decrease in body weight. Cachexia and muscular wasting is secondary to protein degradation by the ubiquitin-proteasome pathway. The aim of this work was to analyze the effect of adjuvant-induced arthritis on the muscle-specific ubiquitin ligases muscle ring finger 1 (MuRF1) and muscle atrophy F-box (MAFbx) as well as on IGF-I and IGF-binding protein-5 (IGFBP-5) gene expression in the skeletal muscle. We also studied whether the synthetic ghrelin receptor agonist, growth hormone releasing peptide-2 (GHRP-2), was able to prevent arthritis-induced changes in the skeletal muscle. Arthritis induced an increase in MuRF1, MAFbx (P < 0.01), and tumor necrosis factor (TNF)-alpha mRNA (P < 0.05) in the skeletal muscle. Arthritis decreased the serum IGF-I and its gene expression in the liver (P < 0.01), whereas it increased IGF-I and IGFBP-5 gene expression in the skeletal muscle (P < 0.01). Administration of GHRP-2 for 8 days prevented the arthritis-induced increase in muscular MuRF1, MAFbx, and TNF-alpha gene expression. GHRP-2 treatment increased the serum concentrations of IGF-I and the IGF-I mRNA in the liver and in the cardiac muscle and decreased muscular IGFBP-5 mRNA both in control and in arthritic rats (P < 0.05). GHRP-2 treatment increased muscular IGF-I mRNA in control rats (P < 0.01), but it did not modify the muscular IGF-I gene expression in arthritic rats. These data indicate that arthritis induces an increase in the activity of the ubiquitin-proteasome proteolytic pathway that is prevented by GHRP-2 administration. The parallel changes in muscular IGFBP-5 and TNF-alpha gene expression with the ubiquitin ligases suggest that they can participate in skeletal muscle alterations during chronic arthritis.     

Elevated plasma free fatty acids decrease basal protein synthesis, but not the anabolic effect of leucine, in skeletal muscle

Elevations in free fatty acids (FFAs) impair glucose uptake in skeletal muscle. However, there is no information pertaining to the effect of elevated circulating lipids on either basal protein synthesis or the anabolic effects of leucine and insulin-like growth factor I (IGF-I). In chronically catheterized conscious rats, the short-term elevation of plasma FFAs by the 5-h infusion of heparin plus Intralipid decreased muscle protein synthesis by approximately 25% under basal conditions. Lipid infusion was associated with a redistribution of eukaryotic initiation factor (eIF)4E from the active eIF4E.eIF4G complex to the inactive eIF4E.4E-BP1 complex. This shift was associated with a decreased phosphorylation of eIF4G but not 4E-BP1. Lipid infusion did not significantly alter either the total amount or phosphorylation state of mTOR, TSC2, S6K1, or the ribosomal protein S6 under basal conditions. In control rats, oral leucine increased muscle protein synthesis. This anabolic response was not impaired by lipid infusion, and no defects in signal transduction pathways regulating translation initiation were detected. In separate rats that received a bolus injection of IGF-I, lipid infusion attenuated the normal redistribution of eIF4E from the active to inactive complex and largely prevented the increased phosphorylation of 4E-BP1, eIF4G, S6K1, and S6. This IGF-I resistance was associated with enhanced Ser(307) phosphorylation of insulin receptor substrate-1 (IRS-1). These data indicate that the short-term elevation of plasma FFAs impairs basal protein synthesis in muscle by altering eIF4E availability, and this defect may be related to impaired phosphorylation of eIF4G, not 4E-BP1. Moreover, hyperlipidemia impairs IGF-I action but does not produce leucine resistance in skeletal muscle.     

Severe diabetes inhibits resistance exercise-induced increase in eukaryotic initiation factor 2B activity.

Rates of protein synthesis are reduced in severely diabetic rats. A potential mechanism through which insulin can stimulate protein synthesis is modulation of the activity of eukaryotic initiation factor 2B (eIF2B). The activity of this factor is elevated after exercise in nondiabetic rats but is markedly lower in skeletal muscle from nonexercised severely diabetic rats. We tested the hypothesis that a failure to increase eIF2B activity after exercise is one potential reason for a failure of severely diabetic rats to increase rates of protein synthesis after resistance exercise. Diabetic (partial pancreatectomy, plasma glucose >475 mg/dl) and nondiabetic male Sprague-Dawley rats (approximately 300 g) performed acute moderate-intensity resistance exercise or remained sedentary. Rates of protein synthesis were higher in nondiabetic rats and increased significantly with exercise, while no elevation was found in severely diabetic rats. The activity of eIF2B was higher (P < 0.05) in exercised nondiabetic than in sedentary nondiabetic rats (0.096 +/- 0.016 and 0.064 +/- 0.02 pmol GDP exchanged/min, respectively), but no difference was observed between sedentary and exercised diabetic rats (0.037 +/- 0.001 and 0.044 +/- 0.008 pmol GDP exchanged/min, respectively), and these activities were lower (P < 0.05) than in nondiabetic animals. These data suggest that severe hypoinsulinemia is associated with an inability to increase eIF2B activity in response to exercise.